快速冷冻与慢速冷冻对复合组织血管内皮生物活性的影响

发布时间：2018-02-20 16:31

本文关键词： 复合组织深低温冷冻冷冻方法内皮细胞活性　出处：《辽宁医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的因复合组织的冷冻存在快速冷冻方法与慢速冷冻方法的争议，所以本实验从这两种冷冻方法对复合组织的血管内皮细胞的影响方面，找到使血管在深低温的冷冻中损伤较小的方法，从而提高断肢（指）冷冻保存后再植手术的成功率。为临床的断肢（指）等复合组织的保存及修复寻找理论基础。方法将35只新西兰大白兔随机分为对照组、快速冷冻12H、快速冷冻3d、快速冷冻7d、慢速冷冻12H、慢速冷冻3d、慢速冷冻7d组，每组5只。①对照组将截断的大白兔10只后肢直接用10%的福尔马林容易固定；快速冷冻12小时、3天、7天组大白兔取后肢每组各10只后肢，直接放入液氮中实行快速冷冻；慢速冷冻组的12小时、3天、7天组大白兔取后肢每组各10只后肢经慢速冷冻后再投入到液氮中，依次保存12小时、3天、7天。所有冷冻组均经37℃水浴快速复温。所有快速复温后的新西兰大白兔后肢均经10%的福尔马林溶液固定。取后肢膝关节至距截肢断端约1cm长度的股动脉血管行HE染色行光镜及免疫组化检查各组血管内皮的病理变化。②对照组大白兔10只前肢取腋动脉约1cm行生物测氧仪测定耗氧量；将快速冷冻及慢速冷冻各组大白兔前肢（每组各10只）经同后肢同样冷冻处理后行37℃快速水浴复温，取大白兔10只前肢取腋动脉约1cm行生物测氧仪测定各实验组耗氧量。结果 ①镜观察对照组内皮细胞较少脱落，各组血管内皮细胞均有不同程度的脱落损伤，经统计分析，实验各组均与对照组存在显著性差异（P0.05）；但是实验组各组间没有显著性差异（P0.05）; ②随着投入液氮后的冷冻时间的增长，不会加重对血管损伤； ③免疫组化检测中,两种冷冻方法血管内皮中均可见血管生长因子呈斑片状的弱表达，与快速冷冻组相比，慢速冷冻组血管活性因子的表达更接近对照组，慢速冷冻组明显高于快速冷冻组（P0.05）。 ④耗氧量的测量结果示：保存12h、3d的慢速冷冻与快速冷冻之间的耗氧量有显著差异（P0.05）。但是慢速冷冻12h、3d组间无差异（P0.05）；快速冷冻12h组、3d组之间也没有差异（P0.05）。结论 1.复合组织经冷冻及复温后血管内皮不同程度损伤； 2.与快速冷冻方法相比，慢速冷冻法对复合组织中的血管内皮细胞影响更少，能更好的保存复合组织中的血管活性。 3.随着投入液氮后的冷冻时间的增长，两种冷冻方法均不会加重对血管损伤；
[Abstract]:objective
Because of the existence of composite tissue freezing quick freezing method and slow freezing method of the dispute, so the effect from the two kinds of composite tissue cryopreservation methods on vascular endothelial cells, to find a way to make small vessels in deep hypothermia frozen injury in limb (finger), so as to improve the success rate of replantation after cryopreservation for the clinical limb (finger) preservation and restoration composite organization to find theoretical basis.
Method
35 New Zealand white rabbits were randomly divided into control group, rapid freezing 12H, quick freezing 3D, quick freezing and slow freezing of 7D, 12H, 3D and slow freezing and slow freezing, 7d group, 5 rats in each group. The control group will cut a total of 10 rabbits with 10% hind Faure Marin easily fixed; frozen 12 hours 3 days, 7 days, the rabbits were hindlimb 10 each hindlimb, directly into the implementation of rapid freezing in liquid nitrogen; slow freezing group 12 hours, 3 days, 7 days of rabbit hindlimb group with 10 rats in each group by hind slow freezing and then into liquid nitrogen, followed by 12 hours of storage for 3 days the 7 day, all groups were frozen. By 37 DEG C water bath rapid rewarming. All rapid rewarming after New Zealand rabbits hind was confirmed by Faure Marin solution 10%. HE fixed femoral artery and posterior knee joint to about 1cm length of amputation stump staining by light microscopy and immunohistochemical examination of blood The pathological changes of endothelium. In the control group, 10 rabbits were taken for forelimb axillary artery about 1cm biological oxygen measuring instrument for determination of oxygen consumption; rapid freezing and slow freezing of each rabbit forelimb (n = 10) with the same freezing treatment by hindlimb was performed in 37 DEG C water bath rapid rewarming, large white rabbits 10 take about 1cm for forelimb axillary artery biological oxygen measuring instrument of oxygen consumption.
Result
(1) the endothelial cells in the control group were less shedding than those in the control group. There were different degrees of shedding injury in each group. After statistical analysis, there was a significant difference between the experimental groups and the control group (P0.05), but there was no significant difference between the experimental group and the control group (P0.05).
(2) the increase of freezing time after the input of liquid nitrogen does not aggravate the damage to blood vessels.
(3) immunohistochemical staining showed that there were plaque like weak expression in vascular endothelial cells of two cryopreservation methods. Compared with the fast freezing group, the expression of vasoactive factors in the slow freezing group was closer to that in the control group, and the slow freezing group was significantly higher than that in the fast freezing group (P0.05).
(4) measurement of oxygen consumption showed that there was a significant difference in oxygen consumption between slow freezing and rapid freezing of 12h, 3D (P0.05). However, there was no difference between slow freezing 12h and 3D groups (P0.05), but there was no difference between fast freezing 12h group and 3D group (P0.05).
conclusion
1. the vascular endothelial cells were damaged in different degrees after freezing and rewarming.
2. compared with the rapid freezing method, the slow freezing method has less effect on the vascular endothelial cells in the composite tissue, and can better preserve the vasoactivity in the composite tissue.
3. with the increase of freezing time after the input of liquid nitrogen, the two cryopreservation methods did not aggravate the damage to blood vessels.

【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.52

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